In recent years, concerns have been raised regarding the presence of harmful proteins, pathogenic proteins, or the like in natural product-derived food materials or feed materials. An example of a harmful protein includes an allergen protein contained in food materials such as buckwheat, wheat, and rice. An example of a pathogenic protein includes an abnormal prion (infectious) contained in materials for edible meat and meat-and-bone meal.
To further illustrate a representative example of a pathogenic protein of recent concern, an abnormal prion is a protein that causes prion disease typified by bovine spongiform encephalopathy (BSE). A normal prion protein is a glyocoprotein that is commonly present in animal brain and on the neural cell membrane surface, and has a molecular weight of approximately thirty-three thousand to thirty-five thousand Daltons(33 to 35 kDa). In contrast, its infectious prion protein form is intracellularly accumulated in the brain (Lait, 76: 571-578, 1996). After entering into an animal body, abnormal prions convert normal prions, which are produced at particular sites in the body, into abnormal prions. This results in the accumulation of the abnormal prions at those particular sites. The accumulation of the abnormal prions in the brain renders the brain spongiform, leading to animal death.
The use of such food materials or feed materials requires detecting and assaying any harmful proteins (e.g., an allergen protein) or pathogenic proteins that are contained in these materials, thereby avoiding the use of the food and feed materials and preventing ingestion of the proteins by humans or animals.
Immunoassay such as ELISA (enzyme-linked immunosorbent assay) or Western blotting (immunoblotting) has here to fore been used to assay natural biological proteins such as prions (abnormal). ELISA is a method performed on a solid phase, wherein an antigen or an antibody is labeled with an enzyme, and the presence of the antibody or the antigen is detected by use of the enzyme activity. For example, ELISA can involve a procedure of binding a Mab 3F4 antibody to a prion immobilized on a microtiter plate and detecting this antibody with a second antibody; the second antibody is coupled to an enzyme which catalyzes a coloring reaction that can be detected (U.S. Pat. No. 4,806,627). Alternatively, Western blotting is a method wherein a protein separated by electrophoresis is immobilized on a hydrophobic membrane, and the protein of interest is detected with an antigen-specific antibody. The detection of an abnormal prion by Western blotting is performed, for instance, by a procedure using a monoclonal anti-prion protein antibody Mab 13A5 (J. Infect. Dis. 154: 518-521, 1986).
However, to detect and assay a prion by a conventional method such as ELISA or Western blotting, the method involves performing in advance, for example, a procedure of digestion and removal of a normal prion from a test sample by treatment with proteinase K. This procedure is for detecting an abnormal prion separately from a normal prion. Western blotting also requires performing electrophoresis. These methods involve complexities and takes much time. Furthermore, in order to achieve the necessary sensitivity, ELISA requires subjecting a sample to denaturation treatment with guanidine thiocyanate after proteinase K treatment. ELISA also requires performing primary denaturation treatment with SDS and a protein concentration procedure by methanol treatment before the deaggregation of the prion protein. In addition, centrifugation must be performed both before the methanol treatment and before the treatment with guanidine thiocyanate. This centrifugation procedure can take much time. In all, these methods involve complicated treatments and therefore present a problem of being unsuitable for testing a large number of samples in a short period of time.
Thus, to improve the problems of ELISA or Western blotting used in the detection and assay of a prion, some other methods have been recently proposed. For example, Japanese Patent Application No. 10-267928 relates to an immuno-PCR method that applies ELISA in detecting an abnormal prion protein with high sensitivity, wherein an anti-prion protein antibody is used and labeled with an arbitrary DNA fragment that is detected by PCR.
Japanese Patent Application No. 2003-130880 relates to a method of immunoassaying an abnormal prion with high sensitivity without performing a time-consuming electrophoresis or centrifugation procedure of the conventional ELISA or Western blotting methods. In this method, a first antibody for inducing an antigen/antibody reaction with an abnormal prion treated with a denaturing agent, or an antigen-binding fragment thereof, is immobilized on magnetic particles and used as an immunoassay reagent. As a result, the method assays the abnormal prion without performing the centrifugation procedure or electrophoresis, and can test a large number of samples in a short time.
Furthermore, Japanese Patent Application No. 2003-215131 relates to a method of analyzing a prion protein by using a mass spectrum, wherein a prion protein in a body fluid sample is allowed to form a covalent bond by reaction with a chemical. Hence, in the presence of a pathogenic prion, at least one additional peak is observed in the mass spectrum.
These methods are modifications of the conventional ELISA or Western blotting methods, although they still must undergo a variety of treatments. Thus, these methods are not necessarily sufficient for conveniently and quickly detecting and assaying an antigenic protein such as a prion. Moreover, these methods are less-than-suitable for automatically or semi-automatically performing treatment steps for detection and assay, and for assaying large amounts of samples.
In contrast, fluorescence correlation spectroscopy (FCS) has been known in recent years as an analysis method that is frequently used particularly in the analysis of molecules derived from organisms. This method can detect and assay, in almost real time, the physical parameters of protein molecules such as number, size, or shape, without undergoing a step of physically separating the sample (Chem. Phys., 4, 390-401, 1974; Biopolymers, 13, 1-27, 1974; Physical Rev. A, 10: 1938-1945,1974; in Topics in Fluorescence Spectroscopy, 1, pp. 337-378, Plenum Press, New York and London, 1991; and R. Rigler, E. S. Elson (Eds.), Fluorescence Correlation Spectroscopy. Theory and Applications, Springer, Berlin, 2001). FCS is practiced by capturing, within an exceedingly small region, the Brownian motions of fluorescence-labeled target molecules in a medium by a laser confocal scanning microscope system. This provides an analysis of the diffusion time from the fluctuation of fluorescence intensity and an assay of the physical parameters of the target molecules (the number and size of the molecules). Thus, FCS serves as an effective means in specifically detecting intermolecular interaction with high sensitivity.
The feature of FCS used in the detection and assay of a protein, or the like, contained in a biological sample is that the concentrations or intermolecular interactions of fluorescence-labeled target molecules contained in a solution can be monitored in almost real time without undergoing a physical separation step. Therefore, a detection system using FCS can avoid a complicated Bound/Free separation step necessary for conventional analysis means (e.g., ELISA) and predominantly used in biomolecule detection systems. This technique can assay large amounts of samples with high sensitivity in a short time and is also suitable for automatic assay.
To detect an antigenic protein or the like by using FCS, a fluorescence-labeled antibody molecule is used, and an antigen/antibody reaction occurs between the fluorescence-labeled antibody and the antigenic protein. Analysis is performed by utilizing the difference in diffusion rate that occurs due to the shapes and molecular weights of the fluorescence-labeled antibody and the fluorescence-labeled antibody/antigenic protein complex. In this context, the diffusion rate (diffusion constant or D) refers to an area where molecules are freely diffused per unit time. The diffusion time (DT or τD) refers to time required for molecules to pass through a determined focal region that depends on the apparatus.
Thus, FCS provides an accurate assay of an antigenic protein or the like in a sample by using the difference in diffusion rates between the labeled antibody and the antigen/antibody complex formed by the labeled antibody and the antigenic protein. FCS could previously detect only the exceedingly limited type of an antigenic protein or the like due to this requirement. Conventional means for solving this problem comprised applying a variety of modifications to an antigen/antibody complex in consideration of the shapes and molecular weights of the antigen and the antibody and providing a significant difference in diffusion rate (Japanese Patent Application No. 2001-272404 and Japanese Patent No. 3517241). However, even if these methods were used, the applicability of FCS was limited regarding which objects could be detected.    Patent Document 1: Japanese Patent Application No. 10-267928    Patent Document 2: Japanese Laid-Open Patent Application No. 2001-272404    Patent Document 3: Japanese Laid-Open Patent Application No. 2003-130880    Patent Document 4: Japanese Laid-Open Patent Application No. 2003-215131    Patent Document 5: Japanese Patent No. 3517241    Non-Patent Document 1: J. Infect. Dis. 154: 518-521, 1986    Non-Patent Document 2: Chem. Phys., 4, 390-401, 1974    Non-Patent Document 3: Biopolymers, 13, 1-27, 1974    Non-Patent Document 4: Physical Rev. A, 10: 1938-1945, 1974    Non-Patent Document 5: in Topics in Fluorescence Spectroscopy, 1, pp. 337-378, Plenum Press, New York and London, 1991    Non-Patent Document 6: R. Rigler, E. S. Elson (Eds.), Fluorescence Correlation Spectroscopy, Theory and Applications, Springer, Berlin,
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention